1. Differentiation of CD4+ T Cells to Th1 Cells Requires MAP Kinase JNK2
Derek D Yang, Dietrich Conze, Alan J Whitmarsh, Tamera Barrett, Roger J Davis, Mercedes Rincón, Richard A Flavell 
Immunity 
Volume 9, Issue 4, Pages (October 1998)
DOI: /S (00)

2. Figure 1 Generation of JNK2-Deficient Mice
(A) The structure of the four murine JNK2 isoforms (JNK2α1, JNK2α2, JNK2β1, and JNK2β2). The alternative splicing of exons that encode sub-domains IX and X (α and β isoforms) and at the COOH terminus (1 and 2 isoforms) is indicated.
(B) The amino acid sequence of mouse JNK2α2 deduced from the sequence of cDNA clones is presented. The alternative sequence of the β isoforms (sub-domains IX and X) and the COOH terminus (1 isoforms) are indicated by the shaded boxes. (#) represents a termination codon.
(C) Strategy for the disruption of the JNK2 gene by homologous recombination. The three hatched boxes are JNK2 exons corresponding to subdomains VIII and IX (amino acid residues 151–229). The tripeptide motif Thr-Pro-Tyr (TPY) includes the dual phosphorylation sites characteristic of the JNK group that is required for protein kinase activity. Restriction enzyme sites are indicated (B, BamHI; H, HpaI; M, MscI; N, NotI; R, EcoRI; and X, XbaI).
Immunity 1998 9, DOI: ( /S (00) )

3. Figure 2
Identification of the Targeted Disruption of the Murine JNK2 Gene
(A) Genomic DNA indicates the presence of all three expected genotypes. EcoRI restricted DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) JNK2-deficient mice was probed with the radiolabeled probe A (Figure 1). The upper band (8.5 kb) corresponds to the wild-type allele (JNK2+) and the lower band (6.0 kb) corresponds to the mutant allele (JNK2−).
(B) Total RNA was isolated from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) knockout mice, and the JNK2 gene and HPRT gene were amplified by RT-PCR.
(C) Western blot analysis of brain, thymus, and CD4+ T cell lysates from wild-type (+/+) and homozygous knockout (−/−) mice. The blots were probed with an antibody to JNK. The bands detected at 46 kDa and 55 kDa correspond to differentially spliced isoforms of JNK1 and JNK2.
Immunity 1998 9, DOI: ( /S (00) )

4. Figure 3
T and B Cell Development Is Similar in Wild-Type and JNK2−/− Mice
Thymocytes (A) or splenocytes (B) from wild-type (WT) and JNK2−/− mice were isolated and stained for CD4 and CD8 and analyzed by flow cytometry. (C) Total lymph node cells were isolated from WT and JNK2−/− mice and stained with anti-CD3 and anti-CD45R/B220 MAbs. (D) Total spleen cells were stimulated with ConA, PMA (5 ng/ml) plus ionomycin (250 ng/ml) (P/I), or medium alone (−). The proliferative response was analyzed by [3H]thymidine incorporation after 3 days. The average of three independent experiments is presented. (E) Total spleen cells were stimulated as described in (D). Supernatants were collected 24 hr following stimulation and assayed for IL-2 production.
Immunity 1998 9, DOI: ( /S (00) )

5. Figure 4
Endogenous JNK Activity and AP1 Transcriptional Activity Are Inversely Related in Th1 Cells from Wild-Type and JNK2−/− Mice
(A) CD4+ T cells were stimulated with ConA, in the presence of APC, and exogenous IL-4 (Th2) or IL-12 (Th1) for 4 days. At day 4, the differentiated Th1 and Th2 cells (5 × 105 cells) were harvested, exhaustively washed, and restimulated with PMA (5 ng/ml) and ionomycin (250 ng/ml). Cells were then harvested and lysed at 0, 30, and 60 min, and cell extracts were assayed for JNK activity. Phosphorylated GST-c-Jun was detected after SDS-PAGE by autoradiography and quantified by PhosphoImager analysis.
(B) Th1 and Th2 CD4+ T cells differentiated for 4 days as described in (A) were lysed, and cell extracts were assayed for JNK protein levels by Western blot analysis.
(C and D) Th1 and Th2 CD4+ T cells differentiated as described in (A) were restimulated (5 × 105 cells) with ConA (C) or immobilized anti-CD3 MAb (D) for different time periods. Cell extracts were assayed for JNK activity as described in (A).
(E) Th1 and Th2 cells (5 × 105 cells) from JNK2+/− × AP1-luciferase and JNK2−/− × AP1-luciferase transgenic mice differentiated as described in (A) were restimulated with ConA for 10 and 24 hr, harvested, and assayed for luciferase activity.
Immunity 1998 9, DOI: ( /S (00) )

6. Figure 5
Analysis of the Cytokines Produced in Effector Th1 and Th2 Cells from Wild-Type and JNK2−/− Mice
(A) FACS profiles represent the CD44 and CD45RB expression in gated CD4+ T cells. Upper gate (CD4+CD44highCD45RBlow) represents memory cells and lower gate (CD44lowCD45RBhigh) represents naive CD4+ T cells.
(B) Th1 and Th2 cells differentiated as described in Figure 4A were restimulated with ConA for 24 hr. Cytokine levels were determined by ELISA.
(C) Cytokine levels produced by Th1 and Th2 cells after restimulation with ConA were determined by ELISA. Values represent average percentages n = 6 of cytokine produced (IL-4 or IFNγ) by JNK2−/− mice versus WT mice.
(D) CD4+ T cells were stimulated with Con A and APC in the absence of exogenous IL-4 or IL-12 for 4 days and restimulated with Con A for 24 hr. Cytokine levels were determined by ELISA.
(E) CD4+ T cells were stimulated with immobilized anti-CD3 MAb (5 μg/ml) or immobilized anti-TCR MAb (10 μg/ml) in the presence or absence of soluble anti-CD28 MAb (1 μg/ml). At day 4, the cells were restimulated with immobilized anti-CD3 MAb (5 μg/ml) or anti-TCR MAb (10 μg/ml) alone for 24 hr. Cytokine levels were determined by ELISA.
Immunity 1998 9, DOI: ( /S (00) )

7. Figure 6
Expression of IFNγ in Effector Th1 and Th2 Cells from Wild-Type and JNK2−/− Mice
(A) Th1 and Th2 cells differentiated as described in Figure 4A were restimulated with Con A. Twenty-four hours after restimulation IFNγ and HPRT mRNA expression was determined in the presence of competitor (200 fg/μl) by competitive RT-PCR as described in Experimental Procedures.
(B) Th1 cells were restimulated with Con A for 24 hr, fixed, permeabilized, and stained with anti-CD4 and anti-IFNγ MAbs, and analyzed by flow cytometry. The dotted line in the histogram represents the negative control and the solid line represents the anti-IFNγ MAb staining in gated CD4+ cells. Values represent the percentage (%) and the mean fluorescence intensity (MFI) of the cells expressing low (Lo) or high (Hi) levels of IFNγ.
Immunity 1998 9, DOI: ( /S (00) )

8. Figure 7
Cytokine Gene Expression in Differentiating Th1 Cells in Wild-Type and JNK2−/− Mice
(A) CD4+ cells were stimulated with Con A in the presence of APC and exogenous IL-12. After 4 days, IFNγ, IL-4, and HPRT gene expression was determined by competitive RT-PCR in the presence of competitor, 100 fg/μl, 33 fg/μl, and 400 fg/μl, respectively.
(B) CD4+ cells were stimulated with ConA in the presence of APC and IL-12 as described in (A). Supernatants were taken at different time points during the differentiation, and IFNγ levels were determined by ELISA.
(C) CD4+ cells were differentiated with Con A in the presence of APC and exogenous IL-4, IL-12, or IL-12 plus IFNγ (200 U/ml) for 4 days and restimulated with Con A for 24 hr. Cytokine levels were determined by ELISA.
(D) CD4+ cells were stimulated with Con A in the presence of APC and IL-12. At day 4, IL-12Rβ2 and HPRT gene expression was determined by semiquantitative RT-PCR.
Immunity 1998 9, DOI: ( /S (00) )